Ion channels expressed in the plasma membrane of excitable tissues (including heart) regulate the function of the tissues. Ion channels can comprise alpha, beta and auxiliary subunits. The alpha subunits are largely responsible for determining overall biophysical properties of the channels, such as ion selectivity, gating and drug sensitivity, whereas beta or auxiliary subunits modify these properties in important ways. Voltage-gated potassium channels comprise four alpha subunits that assemble into a pseudosymmetric array (MacKinnon, 1991), thereby providing the opportunity for heterogeneity by mixing of related subunits to form heterotetrameric channels (Christie et al., 1990; Isacoff et al., 1990). The potential for complexity and heterogeneity increases substantially when beta or auxiliary subunits are also present (England et al., 1995).
Cardiac IKr is a rapidly-inactivating potassium current first identified by its sensitivity to the methanesulfonanilide drug E-4031 (Sanguinetti, M. C. and N. K. Jurkiewicz, 1990). Compared to all other known potassium currents, IKr has a unique functional profile characterized by the suppression of current during depolarization and large, rebounding tail currents produced upon repolarization. Currents are suppressed during depolarization because channels open only briefly and then rapidly inactivate. Upon repolarization, channels recover rapidly from inactivation and revisit the open state. Because deactivation is slow, the channels linger in this highly stable open state and produce the resurgent current that is a hallmark of IKr. Moreover, the sensitivity to E-4031 and other antiarrhythmic drugs is unique to IKr.
Currents with comparable biophysical and pharmacological properties are produced when HERG1, a gene encoding an inwardly rectifying potassium channel that was cloned from human hippocampus (Warmke and Ganetzky, 1994), is transiently expressed in Xenopus oocytes, suggesting that HERG1 is a central component of the channels that give rise to the IKr currents (Sanguinetti et al., 1995; Trudeau et al., 1995). Trudeau, M. C., et al., “HERG, a Human Inward Rectifier in the Voltage-Gated Potassium Channel Family,” Science 269:92 (1995), incorporated by reference as if set forth herein in its entirety, described the HERG gene and also depicted the inwardly rectifying HERG currents and a gating model in the same paper.
Families with a form of inherited (familial) Long QT Syndrome (LQTS-2) have mutations the HERG1 gene (Curran et al., 1995). LQTS-2 is a life-threatening illness characterized by polymorphic ventricular arrhythmias known as torsades de pointes (Roden, 1993). Undiagnosed or untreated, LQTS often leads to sudden death by young adulthood. The expression studies of Trudeau et al. (1995), defining HERG as the primary component underlying IKr, thus explained the underlying cause of LQTS-2 as a loss of IKr.
More clinically prevalent than familial LQTS is an acquired form of the disease caused by block of IKr currents by a surprising variety of drugs, including antiarrhythmic drugs such as dofetilide (Tikosyn®) (Snyders and Chaudhary, 1996), the antihistamines terfenadine (Seldane®) (Roy et al., 1996; Suessbrich et al., 1996) and astemizole (Hismanal®) (Zhou et al., 1999b), the gastric motility drug cisapride (Propulsid®) (Mohammad et al., 1997; Rampe et al., 1997), and cocaine (Zhang S, 2001). An estimated 1-8% of the general public is susceptible to acquired LQTS. Despite their therapeutic value, several of these drugs have been withdrawn from the market because of an unacceptable risk of torsades. As a result, to avoid the risk of torsades and the lost investment associated with withdrawal of a drug from the market, standard pharmaceutical industry practice today dictates that all pharmaceutics in development are screened against cultured cells that express HERG1 in the cell membranes with monitoring for changes in potassium channel behavior. Commercially available HERG-expressing cell lines express only HERG1a channel subunits that assemble into HERG1 channels.
While it is accepted that IKr channels primarily contain HERG 1 subunits, the precise composition of these channels is unknown. The discovery of alternative HERG1a and HERG1b transcripts encoded by the HERG1 gene in human heart (Lees-Miller et al., 1997; London et al., 1997; Kupershmidt et al., 1998; London et al., 1998, each incorporated by reference as if set forth herein in its entirety), raised the possibility that alpha subunits other than HERG1a contribute to the IKr channels.
The proteins encoded by the HERG1a and HERG1b transcripts differ only at their amino termini, as shown in the attached Sequence Listing. The longer amino terminus of HERG1a confers slow deactivation; the shorter amino terminus of HERG1b confers rapid deactivation, relative to HERG1a. When transiently expressed together in a heterologous Xenopus oocyte system, the two subunits assemble to form heteromeric channels that produce currents with unique, intermediate deactivation properties that cannot be explained by the algebraic summation of two homomeric populations of channels. HERG1a DNA and amino acid sequences (SEQ ID NO:1 and 2, respectively) can be found at GenBank Accession No. NM—000238, and HERG1b DNA and amino acid sequences (SEQ ID NO:3 and 4, respectively) can be found at GenBank Accession No. NM—172057). The understanding of the art in this regard is presented in London, B. et al., “Two Isoforms of the Mouse Ether-a-go-go-Related Gene Co-assemble to Form Channels With Properties Similar to the Rapidly Activating Component of the Cardiac Delayed Rectifier K+ Current,” Circ. Res., 81:870 (1997), which is incorporated by reference as if set forth herein in its entirety.
Although HERG1b transcripts have been observed in human heart tissue, until now there was no convincing evidence for the existence in the heart of HERG1b protein, nor was there a consensus as to whether HERG1a and HERG1b channel subunits co-assemble in the heart in vivo. It has heretofore been presumed that HERG channels in cardiac myocytes are uniformly formed of HERG1a subunits and a host of such HERG1a-containing cell lines are available for testing, as described. Even so, the potassium ion channel behavior of HERG1a-containing cell lines does not fully match the behavior of IKr currents observed in cardiac myocytes. Additionally, from the prior work in Xenopus oocytes one cannot predict co-assembly of HERG1a and HERG1b subunits, let alone production of an IKr current, in the membranes of mammalian cells, particularly upon heritable maintenance and expression of HERG1a and HERG1b in such cells. Understanding cardiac IKr physiology and the disease mechanisms of HERG-linked congenital and acquired LQTS necessitates approximating the native state in heterologous systems as closely as possible. It would be desirable to provide improved cell lines for pharmacologic testing, where the improved cell lines mirror the potassium ion channel behavior and subunit composition found in cardiac myocytes.